Recombinant mycobacterial vaccines

ABSTRACT

The present invention relates to recombinant mycobacteria, particularly recombinant M. bovis BCG, which express heterologous DNA encoding a product (protein or polypeptide) of interest, such a protein or polypeptide (e.g., an antigen) against which an immune response is desired, or a cytokine.

RELATED APPLICATIONS

This application is a continuation of application Ser. No. 08/096,027,filed Jul. 22, 1993, now U. S. Pat. No. 5,591,632, which is acontinuation-in-part of Ser. No. 07/711,334, filed Jun. 6, 1991, nowabandoned, which is a continuation-in-part of Ser. No. 07/367,894, filedJun. 19, 1989, now abandoned, said 07/711,334 is a continuation-in-partof PCT/US90/0345 1, filed Jun. 18, 1990 and a continuation-in-part ofPCT/US89/02962, filed Jul. 7, 1989, which are both acontinuation-in-part of Ser. No. 07/361,944, filed Jun. 5, 1989, nowU.S. Pat. No. 5,504,005, which is continuation-in-part of Ser. No.07/223,089, filed Jul. 22, 1998, now abandoned, and acontinuation-in-part of Ser. No. 07/216,390, filed Jul. 7, 1988, nowabandoned, each of which is a continuation-in-part of Ser. No.07/163,546, filed Mar. 3, 1988, now abandoned and PCT/US88/00614, filedFeb. 29, 1988, which is a continuation-in-part of Ser. No. 07/020,451,filed Mar. 2, 1987, now abandoned. All of the above applications areincorporated herein in their entirety.

FUNDING

Work described herein was supported by the United States Public HealthService and the World Health Organization.

BACKGROUND

Several viral and bacterial live recombinant vaccine vehicles are beingdeveloped to produce a new generation of vaccines against a broadspectrum of infectious diseases (Bloom, B. R., Nature 342:115-120(1989)). The human tuberculosis vaccine Mycobacterium bovis bacillusCalmette-Guerin (M. bovis-BCG or BCG) (Calmette et al., Bull. Acad.Natl. Med. (Paris) 91:787-796 (1924)) has features that make it aparticularly attractive live recombinant vaccine vehicle. BCG and othermycobacteria are highly effective adjuvants, and the immune response tomycobacteria has been studied extensively. With nearly 2 billionimmunizations, BCG has a long record of safe use in man (Luelmo, F., Am.Rev. Respir. Dis. 125:70-72 (1982) and Lotte et al., Adv. Tuberc. Res.21:107-193 (1984)). It is one of the few vaccines that can be given atbirth, it engenders long-lived immune responses with only a single dose,and there is a worldwide distribution network with experience in BCGvaccination.

To date, vaccines have been developed which, although effective in manyinstances in inducing immunity against a given pathogen, must beadministered more than once and may be unable to provide protection, ona long-term basis, against a pathogen. In addition, in many cases (e.g.,leprosy, malaria, etc.), an effective vaccine has yet to be developed.

DISCLOSURE OF THE INVENTION

The present invention relates to genetically recombinant (geneticallyengineered) mycobacteria which express DNA of interest which has beenincorporated into the mycobacteria and is expressed extrachromosomally(episomally or autonomously) in the recombinant mycobacteria under thecontrol of a mycobacterial heat shock protein (hsp) promoter, or stressprotein promoter region (e.g., hsp70, hsp60). It particularly relates torecombinant M. bovis-BCG in which DNA of interest is expressedextrachromosomally under the control of a mycobacterial hsp promoter,such as hsp70 and hsp60. DNA of interest is heterologous DNA (i.e., DNAfrom a source other than the mycobacterium into which it is introduced)and is all or a portion of a gene or genes encoding protein(s) orpolypeptide(s) of interest. The protein(s) or polypeptide(s) of interestcan be, for example, those against which an immune response is desired(antigens), enzymes, cytokines, lymphokines and immunopotentiators.

The present invention further relates to vaccines which are geneticallyrecombinant mycobacteria, particularly recombinant BCG, which expressDNA of interest extrachromosomally under the control of a mycobacterialhsp promoter and induce an immune response (e.g., antibody production, Tcell response) in mammals to whom they are administered. A BCG-HIVvaccine which is recombinant BCG which expresses at least oneHIV-encoded polypeptide extrachromosomally under the control of amycobacterial hsp promoter and induces an immune response to thepolypeptide is a specific embodiment of the present invention.

The present invention further relates to a mycobacterial cytokinevaccine which is a recombinant mycobacterium which expresses andsecretes a functional cytokine under the control of a mycobacterial hsppromoter and has been shown to induce endogenous cytokine production,resulting in stimulation of T cells and macrophages. In addition, therecombinant mycobacterium has been shown to be a more potent stimulatorof T cells and macrophages than the mycobacterium alone (wild type). Ina specific embodiment, the recombinant mycobacterium which expresses andsecretes a functional cytokine is recombinant BCG. The recombinant BCGhas been shown to induce endogenous cytokine production to a greaterextent than wild type BCG. The recombinant BCG expressing a cytokineoffer a novel means of enhancing the host (e.g., human and othermammalian) immune response to BCG.

The resulting recombinant mycobacteria are particularly useful asvehicles in which the DNA of interest can be expressed. Such vehiclescan be used, for example, as vaccine vehicles which express apolypeptide or a protein of interest (or more than one polypeptide orprotein), such as an antigen or antigens, for one or more pathogens ofinterest.

The recombinant mycobacteria can also be used as a vehicle forexpression of cytokines, immunopotentiators, enzymes, pharmacologicagents and antitumor agents; expression of a polypeptide or a proteinuseful in producing an anti-fertility vaccine vehicle; or expression ofstress proteins, which can be administered to evoke an immune responseor to induce tolerance in an autoimmune disease (e.g., rheumatoidarthritis). Recombinant mycobacteria can, for example, expressprotein(s) or polypeptide(s) which are growth inhibitors or arecytocidal for tumor cells (e.g., interferon α, β or interleukins 1-7,tumor necrosis factor (TNF) α or β) and, thus, provide the basis for anew strategy for treating certain human cancers (e.g., bladder cancer,melanomas). Pathogens of interest include any virus, retrovirus,microorganism, or other organism or substance (e.g, a toxin or toxoid)which causes disease. The present invention also relates to methods ofvaccinating a host with the recombinant mycobacterium to elicitprotective immunity in the host. The recombinant vaccine can be used toproduce humoral antibody immunity, cellular immunity (including helperand cytotoxic immunity) and/or mucosal or secretory immunity. Inaddition, the present invention relates to use of the polypeptide(s) orprotein(s) such as antigens or cytokines, expressed by the recombinantcultivable mycobacterium as vaccines or as diagnostic reagents.

The vaccine of the subject invention has important advantages overpresently-available vaccines. For example, mycobacteria have adjuvantproperties among the best currently known and, thus, stimulate arecipient's immune system to respond to other antigens with greateffectiveness. This is a particularly valuable aspect of the vaccinebecause it induces cell-mediated immunity and will, thus, be especiallyuseful in providing immunity against pathogens in cases wherecell-mediated immunity appears to be critical for resistance. Second,the mycobacterium stimulates long-term memory or immunity. As a result,a single (one-time) inoculation can be used to produce long-termsensitization to protein antigens. Using the vaccine vehicle of thepresent invention, it is possible to prime long-lasting T cell memory,which stimulates secondary antibody response neutralizing to theinfectious agent or the toxin. This is useful, for example, againsttetanus and diphtheria toxins, pertussis, malaria, influenza, herpesviruses and snake venoms. Recombinant BCG of the present invention whichexpress a cytokine, such as IL-2, are particularly useful because oftheir enhanced immunostimulatory properties (relative to nonrecombinantor wild type BCG). The present invention is, thus, useful to augment theimmunostimulatory properies of BCG in immunization and cancer therapy.Any of a variety of cytokines can be expressed in recombinantmycobacteria, especially recombinant BCG, of the present invention.

BCG in particular has important advantages as a vaccine vehicle inthat: 1) it is the only childhood vaccine currently given at birth; 2)in the past 40 years, it has had a very low incidence of adverseeffects, when given as a vaccine against tuberculosis; and 3) it can beused repeatedly in an individual (e.g., in multiple forms).

A further advantage of BCG in particular, as well as mycobacteria ingeneral, is the large size of its genome (approximately 3×10⁶ bp inlength). Because the genome is large, it is able to accommodate a largeamount of DNA from another source (i.e., DNA of interest) and, thus, canbe used to make a multi-vaccine vehicle (i.e., one carrying DNA ofinterest encoding protective antigens for more than one pathogen).

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1a illustrates the structures of plasmids pY6029, pY6030, andpY6031 which direct expression of HIV1 gag, pol and env polyproteins,respectively, under the control of the mycobacterial hsp70 promoter.

FIG. 1b is a Western blot illustrating the expression of the HIV1 gag,pol, and env gene products by M. bovis BCG electroporated with pY6029,pY6030, or pY6031.

FIG. 2 shows a series of Western blot strips containing HIV proteinswhich were probed with serum from mice vaccinated with wild-type BCG;BCG-HIV gag recombinant cells or BCG-HIV env recombinant cells. Thestrip marked HIV1 was probed with human serum positive for HIVantibodies.

FIGS. 3a-3e is a series of graphs which illustrates the ability of BCGrecombinants to induce cellular immune responses to a foreign pathogen.The levels of α-interferon (pg/ml) produced by spleen cells from miceinoculated with BCG-HIV gag recombinants (FIG. 3a) or nonrecombinant BCG(FIG. 3b) upon stimulation with 5 ug/ml M. tuberculosis purified proteinderivative (PPD, squares), 10 ug/ml each of 5 peptides representing HIV1gag amino acids 256-348 (filled circles), or 10 ug/ml each of 5unrelated peptides (circles) is plotted against time after stimulation.The specific cytotoxic activity of total spleen cells (FIG. 3c), CD8⁺spleen cells (FIG. 3d), or CD4⁺ spleen cells (FIG. 3e) from miceimmunized with BCG-HIV gag recombinants (continuous line) or withwild-type BCG (dashed lines) is also shown.

FIG. 4A is the DNA maps of the relevant portions of (1) HSP60 promoterand polylinker (SEQ ID No: 1) (2) Epitope tag sequence (SEQ ID NO:2),and (3) BCG alpha antigen signal sequence (SEQ ID NO: 3) used inconstruction of IL-2 containing E. coli-BCG shuttle plasmids.

FIG. 4B is a schematic illustration of IL-2 containing plasmidconstructs with restriction sites; P=HSP60 promoter, T=Epitope tag,SS=BCG signal sequence; B=BamHI, E=EcoRI, H=HindIII, C=Cla I and S=SalI.

FIG. 5A is a western blot of BCG pellet (PLT) lysates and BCG culturesupernatants (SN) from RBD recombinants detected with polyclonal rabbitanti-murine IL-2 antibody; Lane 1=recombinant murine IL-2, Lanes2&3=MV261PLT and SN, Lanes 4&5=RBD-2 PLT and SN, Lanes 6&7=RBD-3 PLT andSN, Lanes 8&9=RBD-4 PLT and SN respectively and

FIG. 5B is the identical western blot in FIG. 5a which was reprobed withmmurine monoclonal antibody 12CA5 specific for the influenza epitope tag

FIG. 6 is a graphic representation of expression of biologically activeIL-2 in BCG pellet (PLT) lysates and BCG culture supernatants (SN) fromrat (MAO) and mouse (RBD) recombinant BCG clones.

FIG. 7A is a graphic representation of the time course of splenocyteinterferon-γ production, in response to incubation alone (no Rx), orwith IL-2 (IL-2), wild type BCG (BCG), or recombinant IL-2 secreting BCG(IL2-BCG). 2500 pg/ml of recombinant IL-2 was present at time zero inthe IL2 group. The amount of IL-2 present in the IL2-BCG group includesthat produced by the recombinant BCG and that produced by splenocytes.

FIG. 7B is a graphic representation of the time course of the splenocytetumor necrosis factor production, in response to incubation alone (noRx), or with IL-2 (IL-2), wild type BCG (BCG), or recombinant IL-2secreting BCG (IL2-BCG). 2500 pg/ml of recombinant IL-2 was present attime zero in the IL2 group. The amount of IL-2 present in the IL2-BCGgroup includes that produced by the recombinant BCG and that produced bysplenocytes.

FIG. 7C is a graphic representation of the time course of the splenocyteinterleukin-6 production, in response to incubation alone (no Rx), orwith IL-2 (IL-2), wild type BCG (BCG), or recombinant IL-2 secreting BCG(IL2-BCG). 2500 pg/ml of recombinant IL-2 was present at time zero inthe IL2 group. The amount of IL-2 present in the IL2-BCG group includesthat produced by the recombinant BCG and that produced by splenocytes.

FIG. 7D is a graphic representation of the time course of the totalinterleukin-2 production, in response to incubation alone (no Rx), orwith IL-2 (IL-2), wild type BCG (BCG), or recombinant IL-2 secreting BCG(IL2-BCG). 2500 pg/ml of recombinant IL-2 was present at time zero inthe IL2 group. The amount of IL-2 present in the IL2-BCG group includesthat produced by the recombinant BCG and that produced by splenocytes.Delta-IL2, representing endogenous IL-2 produced by splenocytes wascomputed by subtracting the total IL-2 from the parallel experimentin-which splenocytes were omitted.

FIG. 8A is a graphic representation of interferon-γ production bysplenocytes derived from 3 mouse strains: C3H/HeN, C57BL/6 and BALB/c inresponse to wild type BCG (wtBCG).

FIG. 8B is a graphic representation of interferon-γ production bysplenocytes derived from 3 mouse strains: C3H/HeN, C57BL/6 and BALB/c inresponse to recombinant IL-2 secreting BCG (rBCG).

FIG. 8C is a graphic representation of interferon-γ production bysplenocytes derived from 3 mouse strains: C3H/HeN, C57BL/6 and BALB/c inresponse exogenous IL-2 (25 units=2500 pg) plus wtBCG.

DETAILED DESCRIPTION OF THE INVENTION

Mycobacterium bovis-BCG (bacillus Calmette-Guerin) is an importantclinical tool because of its immunostimulatory properties. Cell wallextracts of BCG have proven to have excellent immune adjuvant activity.Recently developed molecular genetic tools and methods for mycobacteriahave provided the means to introduce foreign genes into BCG (Jacobs, W.R., Jr., et al., Nature 327:532-535 (1987), Snapper, S. B., et al.,Proc. Natl. Acad. Sci. USA 85:6987-6991 (1988), Husson, R. N., et al.,J. Bacteriol 172:519-524) and Martin, C., et al., Nature 245:739-743(1990)). Live BCG is an effective and safe vaccine used worldwide toprevent tuberculosis.

The present invention relates to recombinant mycobacteria, particularlyrecombinant M. bovis BCG, which express heterologous DNA encoding aproduct (protein or polypeptide) of interest, such a protein orpolypeptide (e.g., an antigen) against which an immune response isdesired or a cytokine. Recombinant M. bovis BCG of the present inventionwhich express a cytokine or cytokines have enhanced immunostimulatoryproperties, in comparison with the immunostimulatory properties of M.bovis BCG which does not express a cytokine or cytokines. The enhancedimmunostimulatory properties of recombinant BCG which expresses acytokine or cytokines make that such recombinant BCG particularly usefulas vaccines.

As described herein, it has now been shown that a variety of HIV1polypeptides can be expressed in BCG recombinants under the control of amycobacterial hsp promoter and that the foreign polypeptides produced inBCG induce antibody and T cell responses in mammals to which therecombinant mycobacteria are administered. These results demonstratethat BCG can be used as a live recombinant vaccine vehicle to induceimmune responses to pathogen proteins produced by the bacillus. Inparticular it has been shown that HIV1 polypeptides are expressed in BCGrecombinants under the control of the mycobacterial hsp70 promoter orsimilar mycobacterial promoter and induce immune responses to theencoded proteins in mammals to whom they are administered.

As described herein, three different HIV1 genes have been expressed inrecombinant BCG, using a mycobacterial promoter and translational startsite to direct the expression of the DNA of interest. These regulatoryelements, and particularly the hsp70 promoter, were selected becausethey are among the most powerful in bacteria (Neidhardt, F. C., et al.,In Escherichia coli and Salmonella typhimurium, Cellular and MolecularBiology), F. C. Neidhardt, e al., eds., Washington, DC: American Societyfor Microbiology, 1654 pp (1987)) and because hsp70 synthesis is inducedto very high levels during phagocytosis of some bacteria by macrophage(Buchmeier et al., Science 248:730-732 (1990)). The hsp promoters alsonormally control the expression of proteins that are dominant antigensin the immune response to mycobacterial infection (Young, R. A., Annu.Rev. Immunol. 8:401-420 (1990), Lamb et al., Molec. Biol. and Med.7:311-321 (1990), and Kaufmann, S. H. E., Immunol. Today 11:129-136(1990)). DNA fragments encoding HIV1 gag, pol and env precursors werefused to the mycobacterial hsp70 promoter and translational start siteso that they precisely replaced the hsp70 coding sequences, to producehybrid genes which include HIV1 DNA, the mycobacterial hsp promoter andtranslational start site. The resulting hybrid genes were inserted intothe BCG autonomous replicating plasmid vector pYUB12 (Snapper et al.,Proc. Natl. Acad. Sci. USA 85:6987-6991 (1988)). (FIG. 1a), using knownmethods. The recombinant plasmids were introduced into the Pasteurstrain of M. bovis BCG by electroporation ((Snapper et al., Proc. Natl.Acad. Sci. USA 85:6987-6991 (1988)).

The BCG recombinants produced HIV gag, pol, or env polyproteins whenlysates of the bacteria were examined by Western blotting using serumfrom a HIV-1 positive individual (FIG. 1b). A 52 kD gag polypeptide waspresent in lysates of BCG cells containing the 1.53 kb gag gene, a 92 kDpol polyprotein was found in BCG carrying the 2.76 kb pol gene, and an85 kD env polypeptide was detected in BCG containing the 2.56 kb envgene. The apparent molecular weight of each polyprotein was consistentwith the size predicted by the gene sequence in the absence ofpost-translation modification (gag=512 aa, pol=922 aa, env=856 aa). Bycomparing the signal obtained with known amounts of HIV1 p24 with thatobtained in lysates of BCG-HIV gag recombinants. The amount of fulllength gag precursor protein that accumulates in log-phase cells wasestimated to be approximately 0.1% of total BCG protein. Proteolyticfragments of the HIV1 gag, pol and env precursor proteins were alsodetected. Thus, the total amount of HIV polypeptides that accumulate inthe BCG recombinants may be considerably higher than 0.1% of totalprotein.

When present on the high copy pYUB12 plasmid, the hsp70 promoter is usedconstitutively at high levels in the absence of heat shock, as the HIV1polyproteins did not accumulate to substantially higher levels afterheat shock. In contrast, when integrated as a single copy in the BCGgenome, lower levels of HIV1 gag are produced under the control of thehsp70 promoter at normal temperatures, and heat shock causes an increasein the accumulation of the foreign protein to levels similar to thosefound with the high copy autonomous vector (data not shown).

The ability of BCG recombinants to induce murine immune responses to theforeign proteins produced in BCG was also assessed. BALB/c mice wereinoculated with one primary dose of 5×10⁶ wild-type BCG, 5×10⁶ BCG-HIVgag recombinants, or 5×10⁶ BCG-HIV env recombinant bacilli to studyantibody responses. The mice were inoculated intradermally orintravenously. Mice were bled 5 weeks after inoculation and their serawere used in ELISA and to probe Western blot strips containing HIVproteins (FIG. 2). Although none of the mice vaccinated withnonrecombinant BCG had detectable antibodies to gag or env, 3 mice outof 5 vaccinated with BCG-HIV gag and 1 out of 5 vaccinated with BCG-HIVenv had detectable levels of IgG antibodies reactive against gag (p24and p17) and env (gp160) proteins. All of the mice that producedantibodies against HIV1 proteins had been inoculated with BCGrecombinants intravenously. The levels of HIV-specific antibodies weregenerally low in the antibody-positive mice when tested by ELISA, andsignals were detected when the sera were diluted no more than 1:50 inthe Western blot assay. These results demonstrate that BCG recombinantscan elicit antibody responses to foreign proteins produced by thebacillus.

The ability of BCG recombinants to induce cellular immune responses to aforeign pathogen protein was investigated in mice inoculated withBCG-HIV gag recombinants, by measuring cytokine production and cytotoxicactivity by spleen cells after stimulation with specific antigens.Cytotoxic T lymphocytes (CTL), and to a lesser extent Thl lymphocytes,produce γ-interferon when stimulated with specific antigens or withmitogens, and this response can be measured with clones or in bulkcultures (Mosmann et al., Advances Immunol. 46:111-147 (1989) and Swainet al., J. Immunol. 141:3445-3455 (1988)). Mice inoculated withrecombinant BCG or with BCG-HIV gag were boosted with 5×10⁶ BCG or with5×10⁶ BCG-HIV gag, respectively. Both HIV p24 protein, which is aprocessed segment of the gag polyprotein, and peptides covering theentire gag amino acid sequence were used as stimulating antigens in aγ-interferon production assay. Spleen cells from mice inoculated withBCG-HIV gag recombinants produced substantial levels of γ-interferonwhen stimulated with HIV1 gag peptides, but not when exposed to similarlevels of unrelated polypeptides (FIG. 3a). The level of γ-interferonproduced in response to stimulation with HIV1 gag peptides was similarto that obtained when cells were stimulated with HIV1 p24 (not shown) orwith M. tuberculosis purifed protein derivative (PPD) or concanavalin a(ConA)(FIG. 3a). Spleen cells from mice inoculated with nonrecombinantBCG responded well to PPD and ConA but were nonresponsive to the HIV-gagpeptides (FIG. 3b). The spleen cell populations that producedγ-interferon in response to specific antigens also produced IL-2, asmeasured in a proliferation assay (16) using the IL-2-dependent CTLL-2cell line (not shown). Thl lymphocytes are believed to be the majorsource of IL2 in antigen stimulated spleen cell populations (Mosmann etal., Advances Immunol. 46:111-147 (1989) and Swain et al., J. Immunol.141:3445-3455 (1988)). These results demonstrate that BCG recombinantscan induce murine cell-mediated immune responses to a foreign proteinproduced by the BCG recombinants, and are consistent with theinvolvement of both CTL and Thl lymphocytes.

To investigate further the T cell response to the BCG-HIV gagrecombinant, the antigen specific cytolytic activity of spleen cells andof spleen cells depleted of either CD4 or CD8 cells was measured in a ⁵¹Cr release assay (Nagler-Anderson et al., J. Immunol. 141:3299-3305(1988) and Walker, B. D. in Techniques in HIV Research (ed. Aldovini etal.) 201-210 (Stockton Press, New York, 1990)). Spleen cells from miceimmunized with the BCG-HIV gag recombinant specifically lysed targetcells pulsed with gag petides (FIG. 3c), as did spleen cells depleted ofCD4⁺ cells (FIG. 3d). There was limited specific cytolysis with cellsdepleted of CD8⁺ cells (FIG. 3e); similarly low levels of specificcytolysis were observed when bulk spleen cells were preincubated with amonoclonal antibody that blocks CD8 function. These results indicatethat most of the antigen-specific cytotoxic cells in the spleenpopulation express CD8⁺.

Multiple segments of HIV1 gag protein have been shown to be immunogenicin mice inoculated with the BCG-HIV gag recombinants (Table 1). Sixpools of HIV1 gag peptides, each pool containing five overlapping 25amino-acid peptides, were used to stimulate spleen cells from miceinoculated with BCG-HIV gag recombinants or nonrecombinant BCG. All sixpools of gag peptides stimulated substantial amounts of γ-interferonproduction, albeit to different levels, in spleen cells from miceinjected intravenously with the BCG-HIV gag recombinants. Spleen cellsfrom mice inoculated with nonrecombinant BCG did not respond to any ofthe HIV-gag peptides. Thus, the BCG-HIV gag recombinants consistentlyinduced T cell responses to a variety of epitopes in the foreignprotein.

In another embodiment of the present invention, recombinantmycobacteria, particularly recombinant BCG, containing DNA encoding acytokine (e.g., IL-2) have been produced and shown to produce andsecrete the cytokine in a biologically active form. As described inExample 4, genes encoding IL-2 were inserted into an E. coli-BCG shuttleplasmid under the control of a mycobacterial heat shock protein (hsp)promoter. M. bovis BCG recombinants were constructed that produce andsecrete the mammalian cytokine IL-2 in a biologically active form.Secretion of the active cytokine was accomplished through the combineduse of the BCG hsp60 promoter and a secretion signal sequence derivedfrom the BCG alpha-antigen. The BCG recombinants that secrete IL-2 havebeen shown to stimulate the production of specific lymphokines by mousesplenocyte cultures to a greater extent than wild type BCG stimulatedtheir production, thus demonstrating that BCG recombinants that expressIL-2 and other cytokines are a more potent stimulus of T cells andmacrophages than the wild type BCG and can be used to modify the levelsof specific cytokine production.

An in vitro prototype cytokine expression system for BCG is demonstratedin Example 4. As described, IL-2 encoding sequences are fused with theBCG alpha antigen signal sequence, resulting in expression andextracellular accumulation of biologically active IL-2. Additionalevidence that the signal peptide was responsible for secretion was foundin the Western blot analysis of the BCG recombinants. For each of theBCG recombinants that incorporated the signal sequence, the expressedIL-2 polypeptide appeared to accumulate in BCG cells both with andwithout the signal peptide; in contrast, the size of the single secretedform of IL-2 was consistent with that expected for IL-2 after the signalpeptide has been cleaved. Matsuo et al., previously demonstrated thatHIV epitopes fused to the full length alpha antigen from Mycobacteriumkansasii were secreted with the modified protein after signal peptidecleavage (Matsuo et al., Infect. Immun. 58:4049-4054 (1990)). However,there are no previous reports that the BCG alpha antigen signal peptideitself could direct the extracellular secretion of a full length clonedprotein from BCG.

The selection of the cytokine IL-2 as the first recombinant cytokine tobe tested for secretion from BCG was based on the known central role ofT cell mediated immune responses to BCG infection (Ratliff et al., J.Urol. 17:155-158 (1987)). An in vitro model of immune stimulation wasdeveloped using a mixed population of lymphocytes derived from spleencells to determine if IL-2 secreting BCG would specifically affect aparticular subset of T cells. A modest amount of γ-interferon productionby naive splenocytes in response to BCG was found. IL-4 and IL-5production, however, remained undetectable. This pattern of cytokinesecretion by BCG is consistent with preferential T helper type one(TH-1) activation (Cherwinski et al., J. Exp. Med. 166:1229-1244(1987)). A preferential stimulation of TH-1 cells has been described insplenocytes from C57BL/6 mice previously immunized with BCG orLeishmania Major (Chatelain et al., J. Immunol. 148:1182-1187 (1992))and has been linked to major histocompatibility immune response genes(Huygen et al., Infect. Immun. 60:2880-2886 (1992) and Heinzel et al.,J. Exp. Med. 169:59-72 (1989)).

The most dramatic results from the splenocyte assay were revealed forthe IL-2 secreting BCG recombinant. Both IFN-γ and IL-2 production bysplenocytes were increased approximately 7-8 fold over that produced bynaive splenocytes treated with BCG alone. The effect on IFN-γ productionwas clearly synergistic, since BCG alone, IL-2 alone, nor the simplesummation of their responses was able to generate such high levels. Theproduction of IL-6 and TNF-α were also increased although to a muchlesser extent. A remarkable finding, however, was the capacity of IL-2secreting BCG to increase IFN-γ production from naive splenocytes to alevel well beyond that found for splenocytes treated with BCG alone.This effect was clearly related to the presence of IL-2 as it could bereproduced by the addition of exogenous IL-2 to wild type BCG.Furthermore, neutralizing antibody to IL-2 blocks this response. Asynergistic increase in IFN-γ was shown to occur across 3 differentmouse strains, supporting the concept that the local cytokineenvironment at the time of antigen presentation can significantlyinfluence the direction and amplitude of the immune response. This isparticularly significant for the BALB/c strain which characteristicallyis a poor IFN-γ producer (Heinzel et al., J. Exp. Med. 169:59-72(1989)). These results suggest that this recombinant BCG might beexpected to have enhanced adjuvant and immunostimulatory propertiesabove that found in wild type BCG. The modifications described hereinwhereby BCG is engineered to provide a source of biologically activecytokines represents a novel means to enhance the host immune responseto BCG therapy and study its mechanism of action.

The adjuvant properties of BCG and its cell wall components havepreviously been exploited in experimental vaccines in animals and inman. For example, mixtures of BCG and specific schistosomal antigenshave been used to successfully protect mice in a model ofschistosomiasis (Pierce et al., Proc. Natl. Acad. Sci. USA 85:5678-5682(1988)). An adjuvant/antigen mixture of muramyl dipeptide (MDP) andkilled simian immunodeficiency virus (SIV) have provided partialprotection against SIV infection in macaques (Desrosiers et al., Proc.Natl. Acad. Sci. USA 36:6353-6357 (1989) and Murphey-Corb et al.,Science 246:1293-1297 (1989)); MDP is one of the components ofmycobacterial cell walls that contributes to the adjuvant properties ofBCG. Humans have been vaccinated with mixtures of BCG and killedMycobacterium leprae in large scale trials to assess the efficacy ofthis leprosy vaccine candidate (Bloom, B. R., J. Immunol. 137:i-x(1986)).

As shown herein, recombinant BCG vaccine vehicles can induce immuneresponses to foreign proteins produced by the bacillus, indicating thatBCG can act simultaneously as an adjuvant and as a vehicle to produceand deliver selected antigens to the immune system. The ability toengineer BCG to produce one or more foreign pathogen antigens hasseveral advantages over mixtures of mycobacterial adjuvant and pathogenantigens. Because the antigen continues to be produced by BCGreplicating in vivo, a BCG recombinant may provide a more long-livedimmune response to the pathogen of interest than that provided by thesimple mixture of BCG and antigen. It may be more cost-effective toengineer BCG recombinants than to produce the mixture. Perhaps mostimportantly, the ease with which bacteria can be manipulated geneticallymakes it possible that features of the BCG vaccine vehicle can betailored to maximize the desired immune responses. In addition, asdemonstrated herein, recombinant BCG can be used as a vaccine vehicle toexpress and secrete functional cytokines which induce endogenouscytokine production in cells.

Thus, as described herein, recombinant mycobacterium in which DNA ofinterest is expressed extrachromosomally under the control of amycobacterial hsp promoter have been shown to elicit immune responses tothe proteins produced therein in mammals to which they are administered.They have been shown to elicit an antibody response and to inducecell-mediated immune responses to the protein encoded by the DNA ofinterest.

As also described herein, recombinant mycobacteria, particularlyrecombinant M. bovis BCG, which express a cytokine (e.g., IL-2) or havebeen produced and shown to have augmented or enhanced immunostimulatoryproperties, when compared with M. bovis BCG. which does not express thecytokine.

Recombinant mycobacteria of the present invention can be used, forexample, as vaccines to induce immunity against the pathogenic antigenencoded by the DNA of interest. A pathogen is any virus, microorganism,or other organism or substance (e.g., toxins) which causes disease. Avaccine vehicle useful for immunizing against leprosy can be made.Because of the extraordinary adjuvant activity of mycobacteria, such asBCG, such a vaccine would be effective in producing cell-mediatedimmunity, particularly of a long-term or enduring nature.

Vaccine vehicles which express a protein antigen or antigens frommalaria sporozoites, malaria merozoites, diphtheria toxoid, tetanustoxoid, Leishmania, Salmonella, Mycobacterium africanum, Mycobacteriumintracellulare, Mycobacterium avium, treponema, pertussis, herpes virus,measles virus, mumps, Shigella, Neisseria, Borrelia, rabies, poliovirus, Simian immunodeficiency virus (SIV), snake venom, insect venom orvibrio cholera can also be produced in a manner similar to thatdescribed for HIV1.

The present invention further pertains to vaccine vehicles which expressand secrete cytokines which induce production of endogenous cytokines.In principle, any cytokine gene derived from any source can beintroduced and expressed in BCG. The modified E. coli-BCG shuttlevectors described herein can be used to express and secrete any of avariety of cytokines. The expression of epitope tagged cytokines fromsome of these vectors provides an additional assay for the presence ofthe cytokine. By being able to descriminate between the recombinantcytokine produced by BCG and the cytokine produced by mammalian cells, amore accurate picture of the mechanism of enhanced immunologicalstimulation can be obtained. In this embodiment, the DNA construct(e.g., E. coli-BCG shuttle plasmid) is comprised of the DNA of interest,a promoter and a secretion signal sequence wherein the 5' to 3' order isthe promoter, the secretion signal sequence and the DNA of interest, andthe DNA of interest is under the control of the promoter. The DNAconstruct can additionally comprise an epitope tag for detecting the DNAof interest.

Components of the plasmid introduced into BCG or other mycobacterium(e.g., DNA of interest, hsp promoter and translational start site) canbe obtained from sources in which they naturally occur or can besynthesized, using known techniques, to have substantially the samesequence as the naturally-occurring equivalent. For example, they can beproduced by genetic engineering techniques (e.g., cloning), by thepolymerase chain reaction or synthesized chemically.

It is also possible, using the method of the present invention, toconstruct a multipurpose or multifunctional vaccine (i.e, a singlevaccine vehicle which contains and expresses DNA of interest whichincludes more than one gene, such as a gene encoding a protein antigenfor a different pathogen or toxin and genes encoding a cytokine). Forexample, it is possible to introduce into BCG a gene encoding a proteinantigen for M. lepare, a gene encoding a protein antigen for M.tuberculosis, a gene encoding a protein antigen for Leishmania, a geneencoding a protein antigen for malaria and a gene encoding mammalianIL-2. Administration of this multi-valent vaccine would result instimulation of an immune response to each antigen as well as a morepotent stimulation of T cells and macrophages and provide long-termprotection against leprosy, tuberculosis, leishmaniasis, and malaria.

The recombinant mycobacteria can also be used as an anti-fertility"vaccine" vehicle. For example, mycobacteria containing DNA encodingproteins such as human gonadotropic hormone (HGH) fragments, can be usedas an anti-fertility vaccine and administered as a birth control agent.Vaccine vehicles of the present invention can be used to treat humancancers, such as bladder cancers or melanomas (e.g., by expressinggrowth inhibitors or cytocidal products). In this context, recombinantmycobacteria which contain and express cytokines (e.g., interferon α, βand/or γ, one or more interleukin (interleukins 1-7) and/or TNF α or β)are particularly useful. In another application, recombinantmycobacteria can be used to express stress proteins, either for thepurpose of eliciting a protective immune response (e.g., againstsubsequent or long-term infection) or for the purpose of inducingtolerance in an autoimmune disease (e.g., rheumatoid arthritis). Stressproteins, such as those described in co-pending U.S. patent applicationSer. No. 207,298, entitled Stress Proteins and Uses Therefore, byRichard A. Young and Douglas Young, filed Jun. 15, 1988, can be used forthis purpose. Because of their large genomes (e.g., the BCG genome isabout 3×10⁶ bp long), mycobacteria can accommodate large amounts of DNAof interest, and thus, can serve as multi-purpose vehicles.

Recombinant mycobacteria of the present invention can be administered byknown methods. They can be administered by a variety of routes, such asintradermally or intravenously. They can be administered alone toproduce a desired response, such as an immune response, or can beadministered in combination with the antigen(s) encoded by the DNA ofinterest and/or the killed or attenuated pathogen(s) against which animmune response is desired, in order to enhance or modify the resultingresponse.

The present invention will now be illustrated by the following examples,which are not to be considered limiting in any way.

Example 1

Expression of HIV1 genes in BCG using the Mycobacterial hsp70 Promoterand Ribosome Binding Site.

Construction of pY6029, pY6030 and pY6031.

Three different plasmids containing either the HIV gag, pol or env openreading frames were constructed. DNA fragments containing the codingsequence for HIV1 gag, pol, and env were synthesized by Polymerase ChainReaction (PCR), using the plasmid pHXB2 (Fisher et al., Nature316:262-265 (1985)) as a template, and using oligonucleotide primersspecific for each gene. The upstream primers contained an NcoI site thatoverlapped the AUG translation initiation codon of each HIV1 gene, andthe downstream primer contained an XbaI site immediately after thetranslation stop codon.

To attach the mycobacterial hsp70 promoter and ribosome binding site toeach HIV1 gene, a pUC18 plasmid containing a 1.8 kb segment of the M.tuberculosis hsp70 gene (pY6013) was digested with NcoI and XbaI toremove the hsp70 protein coding sequence (the hsp70 ATG translationinitiation codon sequence overlaps an NcoI site), leaving the ATG and155 bp of upstream hsp70 promoter sequence. The vector DNA containingthe hsp70 promoter was gel purified and ligated to the NcoI/XbaIPCR-derived HIV1 DNA fragments. The HIV1 genes and their associatedmycobacterial regulatory sequences were each transferred from the pUC18vectors to the mycobacterial autonomous replication plasmid vectorpYUB12 (6): EcoRI-XbaI fragments, blunt-ended at the EcoRI site, weregel purified from the first set of plasmids and were inserted betweenthe EcoRV and the XbaI sites of pYUB12 to produce pY6029 (containingHIV1 gag), pY6030 (containing HIV1 pol) and pY6031 (containing HIV1 envshown in FIG. 1a). All manipulations followed previously describedprocedures (Maniatis et al., J. Molecular cloning: a laboratory manual.Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1982)).

Expression of HIV gag, pol or env Polyproteins in M. bovis BCG.

Plasmids pY6029, pY6030 or pY6031 were introduced into the Pasteurstrain of M. bovis BCG by electroporation and cells were plated onMiddlebrook-7HlO agar (Difco) supplemented with albumin dextrosecomplement (Difco), 0.25% tween 20, and ug/ml of kanamycin sulfate.After growth for three weeks at 37° (BCG cells have a doubling time of24 hours), individual colonies were picked and expanded in suspensioncultures in Middlebrook-7H9 broth(Difco) to mid-log phase. Aliquots ofcells (1.5 ml) were harvested by centrifugation and pelleted cells wereresuspended in 50 ul Laemli buffer (Laemli, U. K. Nature 227:680-685(1970)), sonicated with three 20 second pulses using a microtipsonicator, and incubated at 100° D for 4 min. Debris was pelleted bymicrofuge centrifugation for 2 min. and 15 ul of each lysate wassubjected to 10% SDS PAGE. The separated proteins were transferred fromthe gel to a nitrocellulose membrane and probed with human serumpositive for HIV antibodies, followed by ¹²³ I-labeled >Protein A asdescribed in Aldovini, A. and Young R. A., J. Virol., 64:1920-1926(1928). The X-ray film was exposed for 20 hrs., except for lane 5(BCG-env), which was exposed for 60 hrs.

The results of the Western blot analysis of lysates of BCG recombinantsprobed with HIV-positive human serum is shown in FIG. 1b. From left toright, the lanes contain: (1) an HIV1 lysate containing 150 ng p24protein (HIV IIIB); (2) a lysate of wild type M. bovis BCG (BCG); (3) alysate of M. bovis BCG recombinants expressing HIV gag from pY6029(BCG-gag); (4) a lysate of M. bovis BCG recombinants expressing HIV polfrom pY6030 (BCG-pol); and (5) a lysate of M. bovis BCG recombinantsexpressing HIV env from pY6031 (BCG-env).

Example 2

Murine Antibody Response to Vaccination With BCG Recombinants ExpressingHIV1 aag and env.

BALB/c mice were inoculated intradermally or intravenously with oneprimary dose of 5×10⁶ wild-type BCG bacilli, 5×10⁶ BCG-HIV gagrecombinant bacilli or 5×10⁶ BCG-HIV env recombinant bacilli. Mice werebled 5 weeks after inoculation, and the sera were used to probe Westernblot strips of HIV1 proteins (DuPont).

Results of this analysis are shown in FIG. 2. From left to right thelanes were probed with: (1) serum from a mouse injected withnonrecombinant BCG (BCG); (2) serum from a mouse injected with a BCGrecombinant expressing HIV gag (BCG-gag); (3) serum from a mouseinjected with a BCG recombinant exrpessing HIV env (BCG-env); and (4) ahuman serum positive for HIV antibodies (HIV1+). The number of animalsinjected with BCG, or BCG-HIV gag or BCG-HIV env recombinants, and thenumber of animals with positive sera is indicated at the bottom of eachlane.

Example 3

Murine T Cell Response to Inoculation With BCG-HIV gag Recombinants

The ability of BCG recombinants to induce cellular immune responses to aforeign pathogen protein in mice inoculated with BCG-HIV gagrecombinants was investigated by measuring cytokine production andcytotoxic activity by spleen cells after stimulation with specificantigens.

BALB/c mice initially injected with 5×10⁶ nonrecombinant BCG or BCG-HIVgag recombinants were boosted with a similar dose at 4 weeks and then at8 weeks. Spleens were removed at week 9 and cells were cultured andtested for γ-interferon production as described (Wyler et al., J.Immunol. 138:1246-1249 (1987)). Spleen cells were stimulated at aconcentration of 10⁷ cells/ml with antigen or mitogen and supernatantswere removed 48 hrs. and 96 hrs. later. Levels of γ-interferon in thesupernatants were measured in duplicate with a solid-phaseenzyme-immunoassay (Genzyme). Supernatants were diluted where necessaryto obtain γ-interferon values within the linear range of the assay(256-4100 pg/ml); the background cutoff value was 100 pg/ml. A set ofthirty overlapping HIV1 gag peptides covering the entire gag sequence(Ratner et al., Nature 313:277-284 (1985)) was used for antigenicstimulation. Peptides were grouped in 6 pools of 5 peptides each, andused at a concentration of 10 ug/ml per peptide for stimulation.

The results of stimulation with one of the six pools of HIV gagpeptides, representing amino acids 256-348 or with a pool containing 5unrelated peptides as a negative control are shown in FIGS. 3a and 3b.FIGS. 3a and 3b show levels of γ-interferon produced by spleen cellsfrom mice inoculated with BCG-HIV gag recombinants (FIG. 3a) ornonrecombinant BCG (FIG. 3b) after stimulation with 50 ug/ml PPD(squares), 5 ug/ml ConA (triangles), HIV-gag peptides (10 ug/ml each;filled circles) and control peptides (10 ug/ml each; circles). Levels ofγ-interferon production were ascertained for 3 mice inoculatedintravenously with BCG-HIV gag recombinants and 3 mice inoculatedintravenously with nonrecombinant BCG. The results shown in a and b wereobtained from one mouse in each group; the other two mice in each grouphave similar results.

Table 1 shows that multiple segments of HIV1 gag protein are immunogenicin mice inoculated with BCG-HIV gag recombinants. Spleen cells wereobtained from 3 mice inoculated with BCG-HIV gag recombinants, 3 miceinjected with nonrecombinant BCG were pooled, as were cells from theuntreated mice. The results for stimulation of spleen cells from thethree mice injected with BCG-HIV gag recombinants are recordedseparately in Table 1.

The cells (10⁷ /ml) were stimulated with various peptides andγ-interferon levels (pg/ml) in cell supernatants were measured in asolid-phase enzyme-immunoassay (Genzyme) 4 days later (Wyler et al., J.Immunol. 138:1246-1249 (1987)), as described above. Supernatants werediluted 1:10 where necessary to obtain γ-interferon values within thelinear range of the assay (256-4100 pg/ml), and values below 100 pg/mlwere considered negative. Thirty overlapping HIV1 gag peptides (25 aminoacids each and containing 8 overlapping amino acid residues) coveringthe entire gag sequence (Ratner et al., Nature 313:277-284 (1985)) weregrouped in 6 pools of 5 peptides each, and used at a concentration of 10ug/ml per peptide for stimulation. Five unrelated peptides were pooledand used at similar concentrations as a negative control.

To investigate antigen specific cytolytic activity in stimulated spleencells from immunized mice, CD8⁺ and CD4⁺ T cell populations werepurified from total spleen cells by two rounds of negative selection(with complement and with either anti-CD4 or anti-CD8 antibodies) andcharacterized by two color FACS analysis FITC-labeled mAb to CD8(53-6.7, rat IgG2a, Becton Dickinson) and phycoerythrin-conjugated mAbto CD4 (GK1.5, rat IgG2b, Becton Dickinson)! as described(Nagler-Anderson et al., J. Immunol. 141:3299-3305 (1988)). The FACSanalysis demonstrated that the CD8⁺ and CD4⁺ T cell populations werecontaminated less than 1% by cells of the other phenotype. Target celllysis by total spleen or purified CD8⁺ and CD4⁺ T cells was measured bythe standard 4-hr ⁵¹ Cr release assay (Walker, B. D. in Techniques inHIV Research (ed. Aldovini et al) 201-210 (Stockton Press, New York,1990)). Target cells were generated by incubating P815 tumor cells(American Type Culture Collection) with HIV gag peptides at aconcentration of 5 ug/ml per peptide and labelling with ⁵¹ Cr. In eachassay 10⁴ target cells were incubated with varying numbers of effectorcells. Percentage specific ⁵¹ Cr release was calculated from 100X(a-b)/(t-b), where a is ⁵¹ Cr release in the presence of effectorcells, b is the spontaneous release from labeled target cells in theabsence of effector cells and t is the total ⁵¹ Cr content of the targetcells (released by the addition of 1% Nonidet P-40).

The results of this analysis are summarized in FIG. 3, in which thespecific cytotoxic activity of total (FIG. 3c), CD8⁺ (FIG. 3d) and CD4⁺(FIG. 3c) spleen cells from mice immunized with the BCG-HIV gagrecombinant (continuous line), and from mice injected with wild type BCG(dashed line) is shown.

                                      TABLE 1                                     __________________________________________________________________________    Production of γ-interferon (pg/ml) by spleen cells stimulated           with HIV gag peptides.                                                                                            Control                                   Injection                                                                             1-93                                                                              86-178                                                                            171-263                                                                            256-348                                                                            341-433                                                                            426-512                                                                            Peptides                                  __________________________________________________________________________    BCG-HIV gag.sup.(1)                                                                   3500                                                                              800 540  >8200                                                                              800  510  0                                         BCG-HIV gag.sup.(2)                                                                   390 640 280  140  220  4050 0                                         BCG-HIV gag.sup.(3)                                                                   0   0   0    140  140  860  0                                         BCG     0   0   0    0    0    0    0                                         None    0   0   0    0    0    0    0                                         __________________________________________________________________________     Spleen cells were obtained from 3 mice inoculated with BCGHIV gag             recombinatns, 3 mice were injected with nonrecombinant BCG and 3 untreate     mice; spleen cells from mice injected with nonrecombinant BCG were pooled     as were cells from the untreated mice. The cells (10.sup.7 /ml) were          stimulated with varius peptides and interferon levels (pg/ml) in cell         supernatants were measured in a solidphase enzymeimmunoassay (Genzyme) 4      days later (23), as described in FIG. 3 legend. Supernatants were diluted     1:10 where necessary to obtain interferon values within the linear range      of the assay (256-4100 pg/ml), values below 100 pg/ml were considered         negative. Thirty overlapping HIV1 gag peptides (25 amino acids each and       containing 8 overlapping amino acid residues) covering the entire gag         sequence (24) were grouped in 6 pools of 5 peptides each, and used at a       concentration of 10 μg/ml per peptide for stimulation. Five unrelated      peptides were pooled and used at similar concentrations as a negative         control.                                                                 

Example 4

Recombinant BCG Secreting Functional Interleukin IL-2 ModulatesProduction of Splenocytes

Materials and Methods

Oligonucleotide Primers, Plasmid DNAs and Bacterial Strains.

Three sets of paired oligonucleotide primers were utilized in thepolymerse chain reaction (PCR) with appropriate templates to produceinsert DNAs with ends suitable for cloning in the plasmid pMV261. Theoligonucleotide primers were:

for the rat IL-2 gene:

    __________________________________________________________________________    #1: GGCATGGCCAAGGGATCCGCACCCACTTCAAGCCCTGCA (SEQ ID NO: 4);                   #2: CGGAATTCTTACTGAGTCATTGTTGAGATGAT (SEQ ID NO: 5);                          __________________________________________________________________________

for the mouse IL-2 gene:

    __________________________________________________________________________    #3: CAAGGGATCCGCACCCATTCAAGCCCTGCA (SEQ ID NO: 6);                            #4: GCCGGAATTCTTACTGAGTCATTGTTGAGATGAT (SEQ ID NO: 7);                        __________________________________________________________________________

for the alpha antigen signal sequence:

    __________________________________________________________________________    #5: GCCATGCCACAGACGTGAGCCGAAAGATTCGA (SEQ ID NO: 8);                          #6: GCCGGGATCCCGCGCCCGCGGTTGCCGCTCCGCC (SEQ ID NO: 9).                        __________________________________________________________________________

The rat and mouse IL-2 upstream primers #1 (SEQ ID NO: 4) and #3 (SEQ IDNO: 6) respectively were constructed to anneal with the IL-2 codingregions starting at codon 21 thereby excluding their native signalpeptide regions. The BCG alpha antigen downstream primer 14 (SEQ ID NO:7) terminated at the sequence encoding the putative protease cleavagesite ala-gly-ala (Terasaka et al, Complete nucleotide sequence ofimmunogenic protein MPB70 from Mycobacterium bovis BCG. FEMS Lett.58:273-276 (1989)) (FIG. 4A).

The rat IL-2 CDNA containing plasmid pRIL-2.8 was provided by A.McKnight and the mouse IL-2 cDNA plasmid pmut-1 was obtained through theATCC (McKnight et al., Immunogen 30:145-147 (1989) and Yokota et al.,Proc. Natl. Acad. Sci. USA 82:68-72 (1984)). The E. coli/BCG shuttleplasmid pMV261 was kindly provided by C. K. Stover (Stover et al.,Nature 351:456-460 (1991)). The influenza hemagglutinin epitope tagsequence (HA tag) is described in Kolodziej, P. A. and Young, R. A.,Methods Enzymol. 194:508-519 (1991) and had been cloned in the Bgl IIand Bam HI sites of pSP72. (Promega)

E. coli MBM 7070 was obtained from Michael Seidman. Mycobacterium bovisBCG (Pasteur) obtained from ATCC was grown in 7H9 media containing 10%albumin dextrose solution (Difco) and 0.05% tween 80 (Sigma). GenomicBCG DNA was isolated by protease K digestion and phenol/chloroformextraction.

Construction of IL-2 expression vectors and BCG IL-2 recombinantstrains. A schematic representation of the plasmids constructed for thisstudy is given in FIG. 4B. The plasmid pMAO-1 was constructed by placingthe appropriate Bal I/Eco RI digested rat IL-2 PCR insert into thesimilarly restricted parental plasmid, pMV261 (FIG. 4B). The plasmidpMAO-2 was obtained by first cloning the Bam HI/Sal I insert from pMAO-1into the HA tag containing plasmid (FIG. 4A) and then placing theresulting BglII/EcoRI insert into the Bam HI/Eco RI site of pMV261. Theplasmid pMAO-3 was constructed by cloning the Bam I/Bal HI restrictedPCR product encoding the alpha antigen signal sequence into the BalI/Bam HI site of PMAO-1. The plasmid pMAO-4 was produced by replacingthe Bam HI/Eco RI insert of pMAO-3 with the Bgl II/Eco RI insert used inpreparing pMAO-2. A similar set of mouse IL-2 containing plasmids;pRBD-1,2,3 and 4 was produced by replacing the Bam HI/Eco RI rat CDNAinsert in each of the respective pMAO plasmids with the PCR dreived BamHI/Eco RI flanked mouse IL-2 cDNA fragment. All DNA manipulationsfollowed previously described procedures in Maniatis et al., J.Molec.Cloning: a laboratory Manual, Cold Spring Harbor Laboratories, ColdSpring, N.Y. (1982). E. coli MBM 7070was electroporated with the IL-2containing BCG/E. coli shuttle plasmids and selected on kanamycin (30ug/ml) LB agar plates. The correct plasmid structures were confirmed onthe basis of restriction analysis, DNA sequencing and production offunctional IL-2 (see below). E. coli-derived plasmids were then used totransform BCG by electroporation according to published procedures(Snapper et al., Proc. Natl. Acad. Sci. USA 85:6987-6991 (1988)). BCGcolony DNAs were individually tested by PCR for the presence of the IL-2gene and colony lysates were assayed for expression of functional IL-2(see below).

Detection of recombinant IL-2. The expression of recombinant IL-2 in BCGwas examined by Western and by bioassay. Sonicated BCG lysates and BCGculture medium were electrophoresed on a 17-27% acrylamide gel (Daiichi)and transferred to nitrocellulose. After blocking the membrane with a15% solution of powdered skim milk, the membrane was incubated overnightwith the primary antibody, either rabbit anti-mouse IL-2 (CollaborativeResearch) or the mouse monoclonal anti-HA tag antibody 12CA5, at aconcentration of 1 ug/ml (Wilson et al., Cell, 37:76 1984). Peroxidaselabelled goat anti-rabbit or goat anti-mouse IgG antibodies (Pierce)were used with a chemiluninescent substrate (Amersham) for detection.

The presence of biologically active IL-2 in bacterial extracts orextracellular media was determined and quantified colorimetrically in aproliferation assay using the IL-2 dependent T cell line CTLL-2 (Mosmannet al., J. Immun. Methods, 65:55-63 (1983)). Maximal signals generatd inthis assay were similar for either rat or mouse IL-2. E. coli and BCGlysates were obtained by sonication of washed bacterial cells in PBSfollowed by filtration through a 0.22 u filter and dialysis against PBS.No IL-2 inhibitors were found when CTLL-2 cells were incubated withexogenous IL-2 in the presence of extracts prepared from bacteriatransformed with the nonproducer plasmid pMV261. To control fordiffereing growth rates between BCG clones, log-phase BCG were washedand resuspended at an optical density of 0.5 at 600 nm (OD600) in freshmedia. At the end of 48 hours, the OD600 was readjusted to 1.0 bydiluting the BCG cells with fresh media. The amount of IL-2 in 1 ml (1.0OD600˜2-5×10⁷ CFU) of cleared supernatant, or in the pellet derived from1 ml of cells, was then assessed in the proliferation assay.

In vitro spleen cell assay for cytokine production.

Spleens were harvested from 8-12 week old C3H/HeN, C57BL/b or Balb/cmice (Charles River). After mechanical dispersion, the spleen cells wereseparated by Ficoll/hypaque centrifugation at 200 x g, washed, andplaced into RPMI 1640 medium suppliememted with HEPES(N-2-hydroxyethyliperazine-N'-2-ethanesulfonic acid), 10% heatinactivated fetal bovine serum, and 30 ug/ml of kanamycin. Splenocyteassays were performed with either 2 or 4×10⁶ cells/well (1 ml) in thepresence or absence of exogenous murine recombinant IL-2 (Biosource),and either 2×10⁶ CFU MV261 BCG (wild type BCG or wt BCG), or 2×10⁶ CFURBD-4 BCG. Duplicate supernatants were removed at 24 hours and 72 hours,centrifuged and frozen at -70° C. until testing in ELISA assays. Equalspleen cell counts and viabilities were verified prior to final harvestby tryphan blue counting. Equal growth of wt BCG and RBD-4 during the3-day experiment was verified by measurement of optical density at 600nm for parallel wells containing supplemental 0.05% tween 80 to preventbacterial clumping. Cytokine production by spleen cells was measured bycommercial ELISA for murine cytokines, which were used according to themanufacturer's instructions. Kits for the detection of murine IL-4,5,6,and TNF-α were purchased from Endogen. The IFN-γ ELISA was obtained fromGibco/BRL. IL-2 was assayed using a kit from Collaborative Research. Todetect epitope tagged recombinant mouse IL-2, samples were incubated inwells precoated with rabbit anti-mouse IL-2, washed and reincubated withthe murine monoclonal antibody 12CA5 at 1 ug/ml. Bound antibody wasdetected using peroxidase labelled goat anti-mouse IgG (Pierce et al.,Proc. Natl. Acad. Sci. USA 85:5678-5682 (1988)).

Results

Construction of BCG Recombinants Producing IL-2

A variety of E. coli-BCG shuttle plasmids were constructed to permitproduction of IL-2 (FIG. 4). A set of plasmids were constructed in whichthe BCG HSP60 promotor drives the expression of mouse or rat IL-2(pRBD-1 and pMAo-1). To permit differentiation of the BCG-producedrecombinant IL-2 from IL-2 produced by mammalian cells in laterexperiments, a second set of plasmids was generated that incorporate aninfluenza hemagglutinin epitope coding sequence at the 5' end of theIL-2 coding sequence to produce an epitope-tagged IL-2 molecule (pRBD-2and pMAO2). To allow secretion of the recombinant IL-2 molecules, thesecretion signal sequence of the mycobacterial alpha-antigen was addedto the 5' end of the IL-2 coding sequence in a third set of plasmids(pRBD3 and pMAO-3). A fourth set of plasmids contained both the epitopetag and the secretion signal sequence upstream of IL-2 (pRBD-4 andpMAO-4). All constructs containing the IL-2 gene were found to producebiologically active IL-2 in E. coli.

BCG cells were transformed with all of the recombinant plasmids. The BCGtransformation efficiency for both the parental pMV261 and theconstructs containing the alpha antigen signal sequence were on theorder of 10-100 times greater than those IL-2 constructs lacking thesignal sequence. This was a uniform finding occurring in both mouse andrat IL-2 containing constructs and may be due to a selectivedisadvantage caused by the intracellular accumulation of this foreignprotein.

IL-2 Production and Secretion by BCG Transformants

The expression of IL-2 protein by representative BCG recombinants wasassayed by probing Western blots with antibodies directed against IL-2(FIG. 5A) or against the influenza hemagglutinin epitope (FIG. 5B). BCGrecombinants that expressed IL-2 without a secretion signal sequenceaccumulated a single form of IL-2 intracellularly (FIG. 5A, lane 5), butno IL-2 extracellularly (FIG. 5A, lane 4). High and low molecular weightforms of IL-2 accumulated in BCG recombinants that expressed IL-2 linkedto the secretion signal (FIG. 5A, lanes 7-9); only the lower molecularweight form was found in the supernatant, consistent with the cleavageof the signal sequence during secretion (FIG. 5A, lanes 6 and 8). Therecombinant IL-2 proteins that contain the influenza hemagglutininepitope tag can also be visualized with a monoclonal antibody specificfor the tag (FIG. 5B, lanes 5, 8 and 9).

The expression of IL-2 protein by representative BCG recombinants wasalso investigated using an IL-2-dependent proliferation assay (FIG. 6).Most of the biologically active IL-2 produced by clones MAO-1 and RBD-2was located in the pellet while most of the IL-2 product from clonesMAO-3, MAO-4, RBD-3 and RBD-4 was found in the extracellular media. BCGclones expressing IL-2 linked to the alpha antigen signal peptide(MAO-3, MAO-4, RBD-3 and RBD-4) produced significantly more biologicallyactive IL-2 than those clones without the signal peptide (MAO-l andRBD-2). Both mouse and rat IL-2 BCG recombinants expressed similaramounts of bioactive IL-2. The amounts of recombinant mouse IL-2 inpellets and in supernatants were also measured by an ELISA and similarresults were obtained.

Stimulation of Solenocyte Cytokine Production Using BCG-IL-2Recombinants

To evaluate the immunostimulatory properties of IL-2 secreting BCG, theability of BCG recombinants to alter the levels of cytokines IL-2, 4, 5,6, TFN-α and γ-IFN produced by cultured murine spleen cells wasinvestigated (FIG. 7). Splenocytes derived from C3H/HeN mice wereincubated with either no BCG, 25 units/ml of IL-2, MV261 (wt)BCG orRBD-4 BCG. The levels of specific cytokines in the tissue culture mediawere measured by ELISA at 24 and 72 hours after the start of theexperiment.

The data in FIG. 7 shows that no significant basal cytokine expressionwas detected from splenocytes in the absence of BCG or exogenous IL-2.In the IL-2 treated group, there was a modest elevation in IFN-γproduction over the time course of the experiment, but no detectableincreases in other cytokines. By contrast, splenocytes exposed to BCGproduced significant amounts of IL-6, TFN-α and IFN-γ. However, the mostsignificant cytokine production was observed with splenocytes exposed toBCG recombinants secreting IL-2. Substantially higher levels of IFN-γwere produced when spleen cells were exposed to recombinant BCG thanwhen they were exposed to nonrecombinant BCG. Endogenous IL-2production, as calculated by subtracting the total IL-2 in the absenceof splenocytes from the total IL-2 in the presence of splencoytes (FIG.7, delta IL-2), also appeared to increase significantly. Finally, therewas a more modest increase in TFN-α and IL-6 levels when spleen cellswere exposed to recombinant BCG. We did not detect significant amountsof either IL-4 or IL-5 in these splenocyte cultures (lower assay limit100 ug/ml) under any of these experimental conditions.

There is a marked genetic variation in the amount of IFN-γ and IL-2produced by splenocytes derived from mice infected by BCG (Huygen etal., Infect. Immun. 60:2880-2886 (1992)). For example, splenocytes fromBCG-infected C57BL/6 mice produce high levels of IFN-γ and IL-2 whilesplenocytes from BCG-infected BALB/c mice produce low levels of thesetwo cytokines after stimulation in vitro. To determine whether theenhanced immunostimulatory properties of IL-2 secreting BCG were strainindependent, splenocytes were isolated from three different mousestrains, exposed to wild type or recombinant BCG, and the levels ofspecific cytokines in the tissue culture media were measured by ELISA at24 and 72 hours. The results are shown in FIG. 8. As in the previousexperiment, there was very little IFN-γ production by C3H/HeNsplenocytes stimulated with wtBCG (FIG. 8A), but substantial levels wereobserved when the C3H/HeN splenocytes were stimulated with recombinantBCG (rBCG) producing IL-2 (FIG. 8B). Enhanced stimulation was alsoobserved with BALB/c and C57BL/6 splenocytes exposed to rBCG, althoughthe levels of IFN-γ production were somewhat less with BLAB/c andsomewhat greater with C57BL/6. Similar results were obtained ifexogenous IL-2 was added in the presence of wild type BCG (FIG. 8C).There was no detectable IL-4 production in these splenocyte cultures.These results indicate that the enhanced immunostimulatory properties ofIL-2 secreting BCG are not strain dependent.

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 9                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 57 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       ATGGCCAAGACAATTGCGGATCCAGCTGCAGAATTCGAAGCTTATCGATGTCGACGT57                   (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 51 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       AGATCTTCACCATACGACGTCCCAGACTACGCTGGATCCTCTAGAGTCGAC51                         (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 129 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       ATGGCCACAGACGTGAGCCGAAAGATTCGAGCTTGGGGACGCCGATTGATGATCGGCACG60                GCAGCGGCTGTAGTCCTTCCGGGCCTGGTGGGGCTTGCCGGCGGAGCGGCAACCGCGGGC120               GCGGGATCC129                                                                  (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 39 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       GGCATGGCCAAGGGATCCGCACCCACTTCAAGCCCTGCA39                                     (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 32 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       CGGAATTCTTACTGAGTCATTGTTGAGATGAT32                                            (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       CAAGGGATCCGCACCCATTCAAGCCCTGCA30                                              (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       GCCGGAATTCTTACTGAGTCATTGTTGAGATGAT34                                          (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 32 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       GCCATGCCACAGACGTGAGCCGAAAGATTCGA32                                            (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       GCCGGGATCCCGCGCCCGCGGTTGCCGCTCCGCC34                                          __________________________________________________________________________

We claim:
 1. A method of inducing an immune response in a mammalian hostagainst one or more pathogens, comprising administering to the host alive, recombinant mycobacterium, the recombinant mycobacterium havingincorporated therein a plasmid comprising:DNA of interest encoding atleast one protein antigen for each of said pathogens fused to amycobacterial heat shock gene promoter or a mycobacterial stress proteingene promoter and translational start site in such a manner thatexpression of the DNA of interest is under the control of the promoter,wherein the protein antigen is produced by the recombinant mycobacteriumreplicating in the host.
 2. A method of claim 1 wherein the recombinantmycobacterium is selected from the group consisting of:a. Mycobacteriumsmegmatis; b. Mycobacterium bovis-BCG; c. Mycobacterium avium; d.Mycobacterium phlei; e. Mycobacterium fortuitum; f. Mycobacterium lufu;g. Mycobacterium paratuberculosis; h. Mycobacterium habana; i.Mycobacterium scrofulaceiu; j. Mycobacterium intracellulare; k.Mycobacterium tuberculosis; and l. any genetic variants thereof.
 3. Amethod of claim 2 wherein the DNA of interest encodes at least oneprotein antigen selected from the group consisting of,: HIV gag, HIVMol, HIV env, SIV gag, SIV pol and SIV env.
 4. A method of claim 1further comprising co-administering a protein antigen for each of saidpathogens.
 5. A vaccine comprising a live, recombinant mycobacteriumhaving DNA of interest which is expressed extrachromosomally under thecontrol of a mycobacterial heat shock gene promoter or a mycobacterialstress protein gene promoter and an appropriate carrier.